US20100303803A1 - Catenate for immunostimulation - Google Patents

Catenate for immunostimulation Download PDF

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US20100303803A1
US20100303803A1 US12/742,000 US74200008A US2010303803A1 US 20100303803 A1 US20100303803 A1 US 20100303803A1 US 74200008 A US74200008 A US 74200008A US 2010303803 A1 US2010303803 A1 US 2010303803A1
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tumors
molecule
group
composition
molecules
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Matthias Schroff
Burghardt Wittig
Manuel Schmidt
Janine Löhr
Christiane Kleuss
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Mologen AG
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Mologen AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the present invention relates to a multimeric, non-coding nucleic acid molecule for modulation of the activity of the human and animal immune system as well as a method for the manufacture thereof and a vaccine, comprising the multimeric, non-coding nucleic acid molecule, wherein multimeric, non-coding nucleic acid molecules may be understood as non-coding nucleic acid molecules, comprising at least two catenated molecules (dimer) of said non-coding nucleic acid molecules.
  • the cells of the innate immune system recognize pathogens via conserved pathogen associated molecular patterns (PAMP) by germ cell encoded receptors and react immediately.
  • PAMP pathogen associated molecular patterns
  • Different reactions belong to different kinds of cell types like the secretion of cytokines (e.g. IL-1, IL-6, TNF- ⁇ ) and chemokines (e.g.
  • IL-8/CXCL8, MIP-1 ⁇ / ⁇ , MCP-1 the activation of effectors mechanisms (phagocytosis, respiratory discharge, liberation of bactericide substances or lytic granules), the expression of co-stimulatory molecules (CD80, CD86) as well as the enhanced expression of MHC-molecules.
  • effectors mechanisms phagocytosis, respiratory discharge, liberation of bactericide substances or lytic granules
  • co-stimulatory molecules CD80, CD86
  • CpG-oligonucleotides have been used as a new class of immune modulating molecules.
  • Such non-methylated CG-motives can be found in bacterial DNA and represent a “danger signal” for the immune system.
  • PAMP pathogen associated molecular pattern
  • CpG-ODN induce via the cytokines interleukine-12, interferon- ⁇ and tumor necrosis factor-a a T H 1-based immune response.
  • Immune stimulatory nucleic acid sequences comprising said CpG-ODN, have a length of several bases and comprise no open reading frame for the expression of proteins.
  • the ISS represent linear nucleic acid molecules, which ends are open (free hydroxyl- and phosphate groups) or protected by synthetic groups.
  • the strong stimulation of the cellular immune response allows influencing the feedback loops, which will not result in a satisfactory immune activity for the patient without intervention.
  • these monomers made from oligonucleotides have been heated before ligation, receiving uniform molecules out of the oligonucleotides, each consisting of a dumbbell-shaped monomer (compare FIG. 1 of the WO 01/07055). It is known to a person skilled in the art, how to interpret the term monomer consisting of oligonucleotides.
  • monomers may consist out of several molecular elements like oligonucleotide rests, without loosing their character as monomer (e.g. myoglobin is with 153 amino acids a monomer).
  • the monomer is a dumbbell according to FIG. 1 of WO 01/07055.
  • Monomers in the sense of the invention do not designate a structure consisting for instance out of a single base, but does designate a closed dumbbell-shaped form, consisting of nucleotides, which consist their self out of several deoxyribonucleotide rests (compare FIG. 1 or claim 11 of WO 01/07055).
  • Immune stimulation means in the context of the present invention that the mediator and effector cells of the immune system, thus mainly the presently known thymocytes with helper function and the cytotoxic thymocytes, B-cells and so called NK (natural killer)-cells, macrophages and monocytes as well as dendritic cells and their precursors, as well as cell populations with so far not clearly identified functions within the immune system, are stimulated by the use of nucleic acid molecules for proliferation, migration, differentiation or their activity.
  • Immune modulation means, that besides a general stimulation in the above defined sense also the type or character of the immune reaction will be influenced, whether by affecting a beginning or maturing immune reaction or by changing an established reaction with regard to their character.
  • the present invention solves the objective by providing an oligo-respectively multimeric, non-coding nucleic acid molecule. It was completely surprising that dimers, trimers, pentamers or mulitmers of eovalently closed immune stimulatory DNA has an surprisingly improved effect with regard to the molecules known from the state of the art.
  • the invented multimeric molecule can be manufactured by a method, comprising the following steps:
  • a molecule according to the invention can be characterized by its method of manufacture.
  • the method of manufacture serves as definition for the product.
  • the product defined by the method is new with regard to molecules described in the state of the art, like for example in the WO 01/07055.
  • the molecule, which is described and claimed by its way of manufacture is defined by its structural and functional properties, which result from the application of the method of its manufacture for a person skilled in the art.
  • the manufacture of the molecule is very precise using the way of manufacture, because the characterization by structural features is not feasible.
  • the claimed method can be performed successfully, because all necessary declarations for a person skilled in the art are disclosed.
  • the method for manufacture further differentiates from known methods from the state of the art.
  • the molecules according to the invention can also be manufactured by providing 5′-phosphorylated oligodeoxyribonucleotide acids in water, if they are purified with an equivalent method to a polyacrylamide gel electrophoresis. Especially by the combined purification with a HPLC followed by a FPLC. It is known fork a person skilled in the art, that by the combination of several high performance methods like HPLC or FPLC a grade of purification can be reached which is analogue to the grade of a PAGE-purification.
  • a mulitmeric molecule comprising stably catenated monomers and at least 48 nucleotides (2 monomers with 24 nucleotides).
  • the formed catena of molecules does not comprise free 5′- or 3′ ends.
  • the monomers forming via intermolecular catenation the molecule according to the invention are characterized by:
  • a molecule according to the invention comprises at least two monomers and is formed during the above-mentioned synthesis.
  • the monomers are forming intermolecular catena of two, three, four, five or more. This results in the formation of so called di-, tri-, tetra-, penta- or hexamers, so called oligomers as shown in FIG. 1 (picture of gel after separation).
  • a molecule according to the invention can be also defined as catenate.
  • a catenated molecule can be provided for instance, with several monomeric structures been preferably assembled to a catena by their respective loops.
  • a further multimeric structure is the one of the G-quartet.
  • Guanine is able to form due to the orientation of its four H-bridge-binding-sides via guanine-guanine-base pairs a cyclic base quartet with eight H-bridges.
  • a DNA-sequence comprising several sequential guanosine nucleotides, is able to form a higher molecular helical structure, with the guanine bases showing a strong planarity with special staple interaction.
  • number and distribution of guanosine nucleotides within the sequence several G-structures may be formed.
  • a molecule according to the invention is able to modulate the activity of the human or animal immune system better, especially to activate, as molecules from the state of the art.
  • the molecules from the state of the art are the known immune stimulatory nucleic acid sequences, operating as monomeric dumbbell-shaped structures.
  • the most known immune modifying short oligodeoxyribonucleotide acid sequences comprise an unmethylated cytosine-guanosine-motive.
  • a physiological effect of such nucleic acids is also understood as immune modulation respectively modulation of the activity of the immune system within the sense of the invention.
  • the EP 1 196 178 for instance discloses several molecules, consisting of a stem with at least one loop, as they are disclosed for example in the FIGS. 1 , 2 and 3 .
  • Such molecules are monomer structures.
  • the present invention does not comprise such monomers as single molecules.
  • oligomer is used with several different meanings in science.
  • An oligomer may be for instance a longer nucleic acid sequence as well as a structure comprising several of the same or similar molecules formed to a larger assembly.
  • An oligomer within the sense of the invention designates catena of molecules, comprising at least 2-5 monomers forming so called dimers to pentamers. This relates to molecular weights according to FIG. 1 up to 200 kDa.
  • Multimers within the sense of the invention would be for instance several stem-loop-structures according to EP 1 196 178, representing an assembly of several of the same or similar stem-loop-structures to a higher structure (a multimer).
  • multimers are all molecules according to the invention designated which are larger than 200 kDa.
  • the described conditions for the reaction cause during the ligation a stabile intermolecular catenation of the ligations products.
  • a resulting oligomer/multimer will be formed during the synthesis with respect to its confirmation only under the special reaction conditions. It is not possible to manufacture the mulitmers from monomers that have already been formed.
  • the monomer structures forming the multimer are not covalently linked to each other, but they are linked as catenate structures.
  • a formed oligomer (a person skilled in the art realises, that the feature of an oligomer in connection with a multimerization is not the meaning of oligodeoxyribonucleic acid sequence) or a multimer is stabile with respect to heat or denaturing agents, which means that the single molecule structures can not be obtained with simple physical means out of a molecule according to the invention.
  • oligo- and mulitmeric structures have improved and not obvious properties compared to monomeric structures and can be obtained by comparatively simple method steps.
  • the production of assemblies can for instance be performed via centrifugation, gel electrophoresis or column chromatography to show high complex structures, like for instance dimers, pentamers or others, which have compared to single molecule structures improved properties with regard to the modulation of the immune system (compare FIGS. 3 and 4 , 5 ). This results in different forms of immune stimulation in lab organisms or humans.
  • oligodeoxyribonucleotides according to the following characterization are suitable for the method of multimerization.
  • a mulitmer respectively a catenate according to the invention which is characterized by a oligodeoxyribonucleotide sequence used in the method comprising the following sequences:
  • the selection of the preferred sequences leads to molecules, which can be used surprisingly well for the stimulation of the immune system. It is especially preferred, whether the base sequence according to feature c) is comprised in the sequence ggggttac-caccttcattggaaaacgttcttcggggcgtt cttaggtggtaacccctaggggt-taccaccttcattggaaaacgttcttcggggcgttcttaggtggtaaccccta (SEQ ID No. 2) resp.
  • the molecule comprises a partly single stranded, covalently closed chain of the deoxyribonucleotides.
  • oligomeric respectively multimeric structure of the molecule a partly single stranded covalently closed chain of deoxyribonucleotides is responsible for a long term effect of the molecule in the organism in which it is introduced.
  • the molecule comprises the base sequence N 1 N 2 CGN 3 N 4 , wherein N 1 N 2 is an element of the group of GT, GG, GA, AT or AA, N 3 N 4 is an element of the group CT or TT, as well as C deoxycytosine, G deoxyguanosine, A deoxyadenosine and T deoxythymidine.
  • the base sequence N 1 N 2 CGN 3 N 4 is positioned within the single stranded part of the closed chain of deoxyribonucleotides. Especially these preferred molecules show very strong effects during stimulation of the immune systems.
  • the molecule according to the invention thereby comprises not exclusively one deoxyribonucleotide molecule, wherein the deoxyribonucleotide acid molecule,
  • a multimer according to the application may comprise said monomer.
  • a molecule according to the invention may have one or more substitutes bound via covalent binding.
  • substitutes may be e.g. peptides, proteins, saccharides, antigenic structures, lipids, DNA and/or RNA.
  • the invention relates besides the above mentioned steps of a method for the manufacture of a product also to a method for the manufacture of a molecule comprising the following steps:
  • a catenate according to the invention comprises preferably 1+x single components, preferred partly single stranded, dumbbell-shaped covalently closed chains of deoxyribonucleotides,
  • the single components have a stem and a loop, wherein the stem has at least 8 deoxyribonucleotides and the loop at least 4 deoxyribonucleotides and the loop has 1 to 6 CG-motives and x is an element from the set of all natural numbers.
  • the invention relates also to a composition, which comprises at least a molecule according to the invention and a chemotherapeutic. It was surprising that the surprising efficient stimulation of the immune system by a molecule according to the invention could be further improved surprisingly by combining the remedy according to the invention with known chemotherapeutics and using the composition preferably for instance for the treatment of tumours. Although it was known for a person skilled in the art, that monomers according to WO 01/07055 have an immune stimulatory effect and it was further known that chemotherapeutics have an effect on tumours, it was completely surprising that the polymers respectively mulitmers being formed by monomers cause in combination with chemotherapeutics an effect, being beyond aggregation.
  • composition according to the invention functionally interact leading to a synergistic effect.
  • the elements combined in a composition according to the invention have an effect on the same aim to treat pathogens, especially tumours.
  • Each element does not contribute to isolated results within the composition according to the invention, but the interaction between the single elements leads to the surprising effect.
  • a composition according to the invention may be provided as a kit, with a molecule according to the invention and the chemotherapeutics according to the state of the art being provided separately.
  • the at least two components of the kits may be applied simultaneously or time delayed.
  • the application of a composition according to the invention may for instance activate the immune system so that a subsequent application of a chemotherapeutic may have a very good effect.
  • a chemotherapeutic is selected from the group comprising antibodies, alkylating agents, platinum analoga, intercalating agents, antibiotics, mitosis suppresses, taxanes, topoisomerases suppressors, anti-metabolites and/or L-asparaginase, hydroxycarbamide, mitotanes and/or amanitines.
  • the alkylating agents have a very good effect on tumours, inhibiting their growth.
  • intercalating agents are selected from the group comprising:
  • antibiotics are selected from the group comprising:
  • mitoses suppressers are to selected form the group comprising:
  • the toposimerase suppressors are selected from the group comprising:
  • the invention relates further to a kit, comprising the molecule according to the invention and the chemotherapeutic, if applicable together with information about how to combine the content of the kit.
  • the invention relates also—as already described—to a pharmaceutical comprising the molecule according to the invention or the composition if applicable with a pharmaceutical compatible carrier.
  • the invention relates further to the use of the molecule, the composition or the pharmaceutical for the manufacture of a remedy for the modulation of a human or animal immune system or for the modulation of the activity of the mentioned immune system.
  • Modulation of the human or animal immune system each influence on the immune system shall be understood, having the effect that the immune system inhibits tumours or cancer.
  • the modulation of the activity of the immune system can synonymously be understood to this or be described for a person skilled in the art as the known activities of the immune system that are directed against tumours and being surprisingly increased in their activity by remedies according to the invention.
  • the modulation is especially stimulation or an increase of effects of the immune system respectively the immune system itself.
  • a remedy according to the invention can be used in a preferred embodiment to stimulate the T-cell mediated immune response but also the T-cell independent immune response. This process may comprise in a preferred embodiment of the invention a proliferation of B-cells or B-cell activation.
  • the modulation of the activity of the immune system results in stimulation with the effect that cytokines are secreted respectively secretion is enhanced.
  • the molecule according to the invention respectively the composition according to the invention are used as adjuvant in therapeutic or prophylactic vaccination.
  • the remedy according to the invention may be used very efficiently for the treatment of cell growth disorders, wherein in a preferred embodiment the cell growth disorder is a tumour disease.
  • the tumour disease is a disease selected from the group comprising tumours of the ear-nose-throat region, comprising tumors of the inner nose, nasal sinus, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands, and paragangliomas, tumors of the lungs comprising non-parvicellular bronchial carcinomas, parvicellular bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, comprising tumors of the esophagus, stomach, pancreas, liver, gallbladder and biliary tract, small intestine, colon and rectal carcinomas and anal carcinomas, urogenital tumors comprising tumors of the kidneys, ureter, bladder, prostate gland, urethra, penis and testicles, gynecological tumors comprising tumors of the cervix, vagina, vulva, uterine cancer, malignant
  • oligodeoxyribonucleotide (ODN) with the sequence CCTAGGGGTTAC-CACCTTCATTGGAAAACGTTCTTCGGGGCGTTCTTAGGTGGTAACCCCTAGGGGT-TAC-CACCTTCATTGGAAAACGTTCTTCGGGGCGTTCTTAGGTGGTAACC (SEQ ID Nr. 12) were heated for 5 min to 90° C. and subsequently cooled on ice, to enable development of a hairpin structure. Self-complementary overhangs were ligated with a final concentration of 1 mg/ml DNA in the presence of T4-DNA Ligase (0.1 U/ ⁇ g ODN) for 24 h at 37° C. Separation on a 1% agarose gel, each ligation product and after T7 digest, compare FIG. 2 lanes 5 and 6.
  • the oligodeoxyribonucleotide acid sequence CCTAGGGGTTACCACCTTCATTGGAA-AACGTTCTTCGGGGCGTTCTTAGGTGGTAACC (SEQ ID Nr. 13) with a concentration of 1 mg/ml was precipitated with 0.3M sodium-acetate (pH 5.25), 10 mM MgCl 2 and a threefold volume of ethanol abs. After centrifugation (4° C., 13000 rpm) and washing of the pellet for one time with 70% EtOH, the ODN was dried at 50° C. for 10 min.
  • the oligodeoxyribonucleotide acid sequence CTAGGGGTTACCACCTACAAAAAAA-AACGAAATTCGGGGCGAAGGGAGGTGGTAACCC (SEQ ID Nr. 14) with a concentration of 1 mg/ml was precipitated with 0.3M sodium-acetate (pH 5.25), 10 mM MgCI 2 and a threefold volume of ethanol abs. After centrifugation (4° C., 13000 rpm) the ODN was dried at 50° C. for 10 min. The pellet was directly used for ligation (0.5 U/ ⁇ g ODN) and incubated for 60 min at 37° C.
  • a single band can be observed correlating to a single molecule, the monomer (lane 6 after T7 digest). Due to the lower molecular mass the migration is faster and is clearly different with regard to oligomers which are manufactured according to manufacture method b) and which are applied to lanes 1 and 2.
  • oligomers which are manufactured according to manufacture method b) and which are applied to lanes 1 and 2.
  • Several bands can be observed above the band of the monomer representing di- to pentamers. In comparison to this the products manufactured according to method c) are larger and show clearly shorter way of migration, corresponding with larger molecules, compare lanes 3 and 4.
  • Lanes 1 to 3 were loaded with monomer manufactured according to a). Lane 3 shows the starting ODN. One observes a higher molecular structure, which is destroyed by heating at the beginning of the manufacture process. After ligation (lane 2) and T7 digest (lane 1) an enlarged molecules with regard to the starting ODN with a molecular mass of about 50 kDa can be observed corresponding to a monomer.
  • Lane 4 shows again the starting ODN.
  • lane 5 the ligation mixture and lane 6 the ligation after T7 digest.
  • the manufacture is performed according to the conditions as described in b) and c) for the manufacture of oligo- and multimeric molecules. It is clearly visible, that besides single molecules also the desired molecules according to the invention are formed.
  • a band at about 100 kDa can be observed corresponding to a dimer, namely two catenated monomer molecules. The same is observed for tri- and tetramers.
  • TLR9 The ability to stimulate TLR9 was investigated by use of the murine macrophages of the cell line RAW 264.
  • the cells were seeded with 125000 cells/cm 2 and after 16 h the monomeric (as control) and the oligomeric (according to b) an multimeric (according to c) molecules according to the invention were applied. After 7 h of incubation (37° C., 5% CO2) the cells were harvested and measured by fluorescence activated cell sorting (FACS). The results were used to generate a concentration-effect-curve, shown in FIG. 3 .
  • FACS fluorescence activated cell sorting
  • the potency of the molecules according to the invention is increased by a factor of 10 (upper curve) in comparison to the monomeric single molecules (lower curve). Molecules according to the invention have a clearly better effect with less amounts used.
  • the higher potency for immune stimulation can be attributed to a locally higher concentration achieved by the multimeric molecules which can especially in vivo not be achieved by higher doses, e.g. for reasons of the applicable amount.
  • peripheral mononuclear blood cells PBMC
  • the isolated cells PBMC were seeded in multiwell-plates.
  • the first mixture contained not stimulated cells as negative control, the second mixture was stimulated as comparison to the monomers, the third with the oligomeric molecules and the fourth with the multimeric molecules.
  • the secretion of the cytokines interferon-x, and interleukin 6 was determined by ELISA from the cell culture supernatant, compare FIGS. 4 and 5 . According to FIG. 4 the stimulation of PBMCs with the oligo- and multimeric molecules results in a doubling of the INF-gamma secretion in comparison to monomeric single molecules.
  • FIG. 5 shows the IL-6 secretion due to stimulation. While the monomers show stimulation potential comparable to FIG. 4 , the one of the molecules according to the invention is a multiple higher.
  • the nucleic acid sequences are not heated ahead of ligation and have a purification grade comparable to polyacrylamide electrophoresis. It can be provided by itself or by purification via HPLC followed by FPLC. The combination of HPLC and FPLC results in an equivalent purification grade to polyacrylamide electrophoresis. Subsequently the sequences are lyophilised until a dry residue is obtained. A resuspension in a buffer is then made and T4-DNA ligase is added followed from an incubation at 37° C. for 40 minutes. It was surprising, that the obtained concatenates cause an improved immune stimulation in mice. Surprisingly the combination of the of the concatenates according to the invention with chemotherapeutics results in an improved effect.
  • chemotherapeutic antibodies alkylating agents, platinum analoga, intercalating agents, antibiotics, mitosis suppresses, taxanes, topoisomerases suppressors, anti-metabolites and/or L-asparaginase, hydroxycarbamide, mitotanes and/or amanitines may be used.

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Applications Claiming Priority (3)

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EP07075967A EP2058397A1 (de) 2007-11-07 2007-11-07 Multimeres Assemblat zur Immunstimulation
EP07075967.5 2007-11-07
PCT/EP2008/009618 WO2009059805A1 (de) 2007-11-07 2008-11-07 Catenate zur immunstimulation

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CN (1) CN101883854A (pt)
AU (1) AU2008324384A1 (pt)
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CA (1) CA2703983A1 (pt)
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Cited By (6)

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US20130245102A1 (en) * 2010-10-12 2013-09-19 Research Foundation Of The City University Of New York Novel dna templates for small rna production in mammalian cells
GB2542425A (en) * 2015-09-21 2017-03-22 Mologen Ag Means for the treatment of HIV
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
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US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11957788B2 (en) 2014-06-04 2024-04-16 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11578331B2 (en) 2015-09-09 2023-02-14 Gilead Sciences, Inc. Combination comprising immunostimulatory oligonucleotides
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US11583581B2 (en) 2015-09-21 2023-02-21 Gilead Sciences, Inc. Methods of treating a retroviral infection
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)

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CN101883854A (zh) 2010-11-10
KR20100087359A (ko) 2010-08-04
AU2008324384A1 (en) 2009-05-14
MX2010004998A (es) 2010-08-02
EA201000769A1 (ru) 2010-12-30
CA2703983A1 (en) 2009-05-14
EP2207880A1 (de) 2010-07-21
IL205543A0 (en) 2010-12-30
TW200927148A (en) 2009-07-01
JP2011502492A (ja) 2011-01-27

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